Patent Application
20050014816
The present invention relates to novel thiophene amino acid derivatives, for interacting with metalloproteases, and more specifically with macrophage metalloelastase (MMP-12), and for the prevention and treatment of respiratory pathologies such as chronic obstructive bronchopneumopathy (COPD), emphysema, chronic bronchitis, chronic pulmonary inflammation, asthma, cystic fibrosis, acute respiratory distress syndrome (ARDS), respiratory allergies including allergic rhinitis, and also diseases associated with the production of TNFα including severe fibrotic pulmonary disease, pulmonary sarcoidosis and silicosis. The compounds of the present invention also show inhibitory activity on metalloprotease-13 (MMP-13), making them useful for the treatment of pathologies involving this enzyme, such as cancer, osteoporosis, osteoarthritis, arthritis, rheumatoid arthritis, atherosclerosis, multiple sclerosis and cardiac insufficiency. In addition, the invention relates-to methods and processes concerning the same, and to compositions containing the same.
The compounds of the present invention also show, to a lesser extent, inhibitory activity on metalloprotease-13 (MMP-13), making them potentially useful for the treatment of pathologies involving this enzyme, such as cancer, osteoporosis, osteoarthritis, arthritis, rheumatoid arthritis, atherosclerosis, multiple sclerosis and cardiac insufficiency.
Metalloproteases (MMPs) are a large family of proteases that degrade the extracellular matrix and are secreted especially by mesenchymal cells, macrophages and polymorphonuclear leukocytes. Metalloproteases are classified into several subfamilies depending on their primary structure and their specificity. These families especially include collagenases (MMP-1, MMP-8 and MMP-13), stromelysins (MMP-3 and MMP-10), gelatinases (MMP-2 and MMP-9), matrilysin (MMP-7), macrophage metalloelastase (MMP-12) and also MMPs of membrane-bound type (MMP-14, MMP-15, MMP-16 and MMP-17).
MMPs are zinc metalloproteases that have the ability to degrade virtually all the components of the extracellular matrix, ie the interstitium and the basal membranes. Increased synthesis of these enzymes is found in many destructive diseases (inflammatory arthritis, atherosclerosis, tumoral invasion and angiogenesis). MMPs (in particular those with powerful elastolytic activity) are involved in the physiopathology of asthma and chronic obstructive bronchopneumopathies including tobacco-related pulmonary emphysema (COPD).
Human macrophage elastase (HME or MMP-12) shows all the characteristics of the other MMPs. It degrades many macromolecules of the extracellular matrix (gelatin, fibronectin and laminin) and especially elastin. MMP-12 is not synthesized by the circulating monocytes but solely by macrophages or monocytes differentiated in vitro into macrophages. The pathology of emphysema is characterized by destruction of the elastin present in the walls of the pulmonary alveolae. Demonstration of the increase in the level of MMP-12 during the manifestation of this pathology thus suggests a predominant role of this enzyme in the occurrence and development of this disease. Similarly, studies have demonstrated the absence of development of emphysema in MMP-12-deficient mice, these mice being exposed for a long time to cigarette smoke (Science 1997, 277, 2002-2004). More recently, also using MMP-12-deficient mice, in an asthma model, a group has suggested the involvement of MMP-12 in the development of chronic asthma (FASEB, 2002, 16, A590). These results clearly demonstrate that human macrophage elastase (MMP-12) inhibitors might be very useful for preventing and treating chronic respiratory pathologies such as chronic obstructive pulmonary bronchitis (COPD), emphysema, chronic bronchitis and chronic pulmonary inflammation, and also respiratory pathologies caused by an inflammation phenomenon, such as asthma, mucoviscidosis, acute respiratory distress syndrome (ARDS), repiratory allergies including allergic rhinitis and also diseases associated with the production of TNFα including severe fibrotic pulmonary disease, pulmonary sarcoidosis and silicosis.
All metalloproteases have a catalytic domain consisting of 162 to 173 amino acids containing the active site of the enzyme. A Zn2+ ion is present in the active site, to which it is bound via histidine residues. This site is one of the preferred points of attachment of synthetic MMP inhibitors, since it especially allows the creation of a stable, powerful chelation centre that is readily accessible to small molecules. Thus, all the powerful inhibitors described in the literature contain a chemical function such as a hydroxamic acid allowing chelation between the zinc atom of the catalytic site of the metalloprotease and the said inhibitor. This chelation ensures blockage of the active site and results in inhibition of the said enzyme.
One of the major problems of inhibition of this type is the absence of selectivity or the low degree of selectivity, since all MMPs contain a zinc ion in their active site. The second problem associated with these powerful but generally poorly selective inhibitors is the toxicity associated with the presence of a chemical function such as a hydroxamic acid.
One of the objects of the invention is thus to provide novel compounds that have inhibitory properties on type 12 metalloprotease (MMP-12). A solution has been found by producing novel thiophene amino acid derivatives, and also by using the said compounds in pharmaceutical compositions that can be used in the prevention and treatment of pathologies associated with an inhibition of MMP-12.
Several scientific articles and patent applications describe compounds comprising a central thiophene unit. Among this literature, mention may be made of patent application WO 98/23605, which describes thien-2-ylcarboxamide derivatives substituted in position 4 with a cyclic system and in position 5 with a trifluoromethyl group. These compounds are claimed for their bactericidal and fungicidal activity. Patent application WO 96/16954 also describes compounds optionally comprising a 4-aryl-thien-2-ylcarboxamide system in which the amide function may be substituted with a phenyl group, which are useful for their antifungal properties.
None of these documents describes or suggests for these compounds inhibitory activity on MMP-12 or mixed MMP-12-13 inhibitory activity and a potential use of this type of product in the treatment of respiratory pathologies, or of pathologies of inflammatory type, which is a novel property of the compounds claimed by the applicant.
The invention provides compounds of Formula (I),
stereoisomers thereof, or pharmaceutically acceptable salts of said compounds or stereoisomers, wherein R1, R2, m, p, q, R7 and R8 are as defined below, as well as compositions comprising the same, processes for making the same, and methods of using the same to treat a variety of diseases, including, those requiring interaction with metalloproteases, and more specifically with macrophage metalloelastase (MMP-12), and for the prevention and treatment of respiratory pathologies such as chronic obstructive bronchopneumopathy (COPD), emphysema, chronic bronchitis, chronic pulmonary inflammation, asthma, cystic fibrosis, acute respiratory distress syndrome (ARDS), respiratory allergies including allergic rhinitis, and also diseases associated with the production of TNFα including severe fibrotic pulmonary disease, pulmonary sarcoidosis and silicosis. The compounds of the present invention also show inhibitory activity on metalloprotease-13 (MMP-13), making them useful for the treatment of pathologies involving this enzyme, such as cancer, osteoporosis, osteoarthritis, arthritis, rheumatoid arthritis, atherosclerosis, multiple sclerosis and cardiac insufficiency.
The invention provides compounds of Formula (I),
in which:
According to one advantageous variant of the invention, the preferred compounds of the invention are the compounds of formula (I) in which:
According to one particularly advantageous variant of the invention, the phenyl group in the compounds of formula (1) is substituted with a group R1 as defined in the formula (1), located in the para position.
The groups R1 that are preferred according to the invention are groups selected from trifluoromethoxy, 4-acetylphenyl, 4-pyridyl, 3-pyridyl, N-pyrrolidinyl, 1-methylpyrrol-3-yl, 3,6-dihydro-2H-pyrid-1-yl, cyclohexyl, 2-hydroxy-4-pyridyl and 2-hydroxyphenyl.
Very advantageously, R1 represents a group selected from trifluoromethoxy, 4-acetylphenyl, cyclohexyl and 4-pyridyl.
Preferably, q is an integer equal to zero.
According to one advantageous variant of the invention, m is an integer selected from zero and one.
Similarly, for the preferred compounds of the invention, p is advantageously an integer selected from zero and one.
The group R7 that is preferred according to the invention is the phenyl group.
The groups R8 that are preferred according to the invention are groups selected from carboxyl and aminocarbonyl.
The isomers, and also the addition salts with a pharmaceutically acceptable acid or base, of the variants and the preferred compounds form an integral part of the invention.
The invention also relates to the pharmaceutically acceptable salts of the compounds of formula (I). A review of pharmaceutically acceptable salts is described especially in J. Pharm. Sci., 1977, 66, 1-19.
The expression “pharmaceutically acceptable acids” means non-toxic organic or mineral acids. Among the pharmaceutically acceptable acids that may be mentioned, without any limitation, are hydrochloric acid, hydrobromic acid, sulphuric acid, phosphonic acid, nitric acid, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, oxalic acid, methanesulphonic acid, camphoric acid, benzoic acid, toluenesulphonic acid, etc.
The expression “pharmaceutically acceptable bases” means non-toxic organic or mineral bases.
Among the pharmaceutically acceptable bases that may be mentioned, without any limitation, are sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, triethylamine, tert-butylamine, 2-diethylaminoethanol, ethanolamine, ethylenediaamine, dibenzylethylenediamine, piperidine, pyrrolidine, morpholine, piperazine, benzylamine, arginine, lysine, histidine, glucamine, glucosamine, quaternary ammonium hydroxides, etc.
In general, the expression “isomers of the compounds of the invention” means optical isomers such as enantiomers and diastereoisomers. More particularly, the pure enantiomeric forms of the compounds of the invention may be separated from mixtures of enantiomers that are reacted with a releasable agent for resolving the racemic mixtures, the said agent itself existing in the form of a pure enantiomer, allowing the corresponding diastereoisomers to be obtained. These diastereoisomers are then separated according to the separation techniques that are well known to those skilled in the art, such as crystallization or chromatography, and the resolving agent is then removed using the standard techniques of organic chemistry, to produce a pure enantiomer. In another manner, the pure enantiomeric forms of the compounds of the invention may be separated by chromatography on a chiral column.
The compounds of the invention that are present in the form of a mixture of diastereoisomers are isolated in pure form by using standard separation techniques such as chromatographies.
In certain particular cases, the process for separating the compounds of the invention may lead to the predominant formation of one enantiomer or one diastereoisomer relative to the other.
The invention also covers the process for preparing the compounds of formula (I). More particularly, the compounds of formula (I) may be obtained from the compounds of formula (II):
The compounds of formulae (II), (V), (VII), (IX), (X) and (Xa) are either commercial compounds or are obtained according to known methods of organic synthesis that are readily available and comprehensibles to those skilled in the art.
According to one variant of the invention, the compounds of formula (1) may also be obtained via a second preparation process characterized in that the starting material used is a compound of formula (II):
On account of their pharmacological properties as MMP-12 inhibitors, the compounds of the present invention are useful in the prevention and treatment of respiratory pathologies such as chronic obstructive bronchopneumopathy (COPD), emphysema, chronic bronchitis, chronic pulmonary inflammation, asthma, mucoviscidosis, acute respiratory distress syndrome (ARDS), respiratory allergies including allergic rhinitis, and also diseases associated with the production of TNFα, including severe fibrotic pulmonary disease, pulmonary sarcoidosis and silicosis. The compounds of the present invention also show, to a lesser extent, inhibitory activity on metalloprotease-13 (MMP-13), making them potentially useful for the treatment of pathologies involving this enzyme, such as cancer, osteoporosis, osteoarthritis, arthritis, rheumatoid arthritis, atherosclerosis, multiple sclerosis and cardiac insufficiency.
Advantageously, the compounds of the present invention are useful for preventing and treating chronic obstructive bronchopneumopathy, emphysema and chronic bronchitis.
More particularly, the compounds of the present invention are useful for treating tobacco-related emphysema.
According to one variant of the invention, the compounds of formula (I) are useful for preventing and treating asthma.
The subject of the present invention is also pharmaceutical compositions containing as active principle at least one compound of formula (I), an isomer thereof or an addition salt thereof with a pharmaceutically acceptable acid or base, alone or in combination with one or more inert, non-toxic, pharmaceutically acceptable excipients or vehicles.
Among the pharmaceutical compositions according to the invention, mention will be made more particularly of those that are suitable for oral, parenteral (intravenous, intramuscular or subcutaneous), percutaneous or transcutaneous, intravaginal, rectal, nasal, perlingual or respiratory administration.
The pharmaceutical compositions according to the invention for parenteral injections especially comprise aqueous and non-aqueous sterile solutions, dispersions, suspensions or emulsions and also sterile powders to reconstitute injectable solutions or dispersions.
The pharmaceutical compositions according to the invention for solid oral administration especially comprise simple or sugar-coated tablets, sublingual tablets, sachets, gel capsules and granules, and, for oral, nasal or buccal liquid administration, especially comprise emulsions, solutions, suspensions, drops, syrups and aerosols.
The pharmaceutical compositions according to the invention for administration via the respiratory route especially comprise compositions in the form of solutions for aerosols or powders for inhalers. When the compositions are aerosols, for the use of liquid aerosols, the compositions may be stable sterile solutions or solid compositions dissolved at the time of use in apyrogenic sterile water, in physiological saline or in any other pharmaceutically acceptable vehicle. For use in the form of dry aerosols intended to be inhaled directly, the active principle is optionally finely divided or micronized, and combined with an inert, solid, water-soluble diluent or vehicle.
The pharmaceutical compositions for rectal administration are preferably suppositories, and those for percutaneous or transcutaneous administration especially comprise powders, aerosols, creams, ointments, gels and patches.
The pharmaceutical compositions mentioned above illustrate the invention but do not limit it in any way.
Among the inert, non-toxic, pharmaceutically acceptable excipients or vehicles that may be mentioned, as a guide and with no limitation, are diluents, solvents, preserving agents, wetting agents, emulsifiers, dispersants, binders, swelling agents, crumbling agents, retardants, lubricants, absorbing agents, suspension agents, colorants, flavourings, etc.
The practical dosage varies according to the age and weight of the patient, the route of administration, the pharmaceutical composition used, the nature and severity of the complaint, and the possible taking of associated treatments. The dosage ranges from 1 mg to 1000 mg in one or more dosage intakes per day.
The examples that follow illustrate the invention but do not limit it in any way.
The starting materials used are commercial products or products prepared according to known procedures from commercial compounds or compounds known to those skilled in the art. The various preparations give synthetic intermediates that are useful for preparing the compounds of the invention.
The structures of the compounds described in the examples and in the preparations were determined according to the usual spectrophotometric techniques (infrared (1R), nuclear magnetic resonance (NMR), mass spectrometry (MS) including electron spray (ES) mass spectrometry, etc.) and the purity was determined by high performance liquid chromatography (HPLC).
Abbreviations used in the procedures:
84.9 ml (2.1 equivalents) of a 2.0M solution of potassium phosphate and 2.8 g (0.03 equivalent) of tetrakis(triphenylphosphine)palladium(0) are added to a solution of 12.3 g of 4-bromothiophene-2-carbaldehyde and 20.0 g of [4-(trifluoromethoxy)phenyl]-boronic acid (1.2 equivalents) in 70 ml of degassed DME. The reaction medium is stirred for 3 hours at 80° C. and then concentrated under reduced pressure. The residue obtained is taken up in ethyl acetate. The solution is then filtered through Celite, washed with water, dried over sodium sulphate, filtered and then concentrated under reduced pressure. Chromatography of the residue on silica gel (9/1 cyclohexane/ethyl acetate) allows 15.05 g of the expected product to be isolated.
Yield: 68%
1H NMR (CDCl3) δ (Ppm): 10.0 (s, 1H), 8.0 (s, 1H), 7.80 (s, 1H), 7.55 (m, 2H), 7.25 (m, 2H)
Stage 2: 4-[4-(Trifluoromethoxy)phenyl]thiophene-2-carboxylic acid
37.6 g (4 equivalents) of silver nitrate and 44.2 ml (8 equivalents) of aqueous 1.0M sodium hydroxide solution are added to a solution of 15.05 g of the compound obtained in stage 1 in 200 ml of ethanol. The reaction medium is stirred for 2 hours at 40° C., then filtered through Celite and concentrated under reduced pressure. The aqueous phase is washed with aqueous 1.0M hydrochloric acid solution, extracted with ethyl acetate, dried over sodium sulphate, filtered and concentrated under reduced pressure, to give 15.514 g of a beige-coloured powder corresponding to the expected product.
Yield: 97.4%
MS: MH−287
Preparation 2: (2S)-2-Amino-N-cyclohexyl-2-phenylacetamide
Stage 1: (2S)-tert-Butoxy-2-(cyclohexylamino)-2-oxo-1-phenylethyl carbamate
0.164 ml of cyclohexylamine (1.2 equivalents), 0.548g of O-[(ethoxycarbonyl)-cyanomethyleneamino]-N,N,N′,N′-tetramethyluronium (TOTU) and 830 μl of N-ethyl-N,N-diisopropylamine are added to a solution of 300 mg of (2S)-[(tert-butoxycarbonyl)amino](phenyl)acetic acid in 5 ml of anhydrous dimethylformamide. The reaction medium is stirred for 3 hours at room temperature and then concentrated under reduced pressure. The residue obtained is dissolved in ethyl acetate (30 ml), washed with water (30 ml), dried over sodium sulphate, filtered and concentrated under reduced pressure. Chromatography of the residue on silica gel (70/30: cyclohexane/ethyl acetate) allows 258 mg of the expected product to be isolated.
Yield: 65%
MS: MH+333
Stage 2: (2S)-2-Amino-N-cyclohexyl-2-phenylacetamide
0.297 ml of trifluoroacetic acid is added at 0° C. to a solution of 258 mg of the product obtained in stage 1 in 4 ml of anhydrous dichloromethane. The reaction medium is stirred for 17 hours at room temperature, washed with water (30 ml) and then with saturated sodium hydrogen carbonate solution (30 ml), dried over sodium sulphate, filtered and concentrated under reduced pressure. Recrystallization of the residue from diisopropyl ether allows 219 mg of the expected product to be isolated.
Yield: 99%
1H NMR (CDCl3) δ (Ppm): 7.30 (m, 5H), 6.90 (m, 1H), 4.50 (m, 1H), 4.10 (m, 2H), 3.80 (m, 1H), 2.50 (m, 1H), 1.60 (m, 12H)
MS: MH+233
HPLC: 97.1%
Preparation 3: Ethyl (3S)-3-amino-4-phenylbutanoate
1.0 ml of concentrated sulphuric acid (10 volumes) is added to a solution of 0.5 g of (3S)-3-[(tert-butoxycarbonyl)amino]-4-phenylbutanoic acid in 10 ml of ethanol. The reaction medium is stirred for 17 hours at reflux. The solution is then concentrated under reduced pressure and basified with saturated sodium hydrogen carbonate solution to pH 8. The solution obtained is extracted with ethyl acetate and the organic phase is dried over sodium sulphate and then filtered to give 0.286 g of the expected product after evaporation under reduced pressure.
Yield: 77%
1H NMR (DMSO) δ (ppm): 7.30 (m, 2H), 7.20 (m, 3H), 4.0 (q, 2H), 3.20 (m, 1H), 2.70 (m, 2H), 2.30 (m, 2H), 1.50 (s, 2H), 1.10 (t, 3H)
MS: MH+208
HPLC: 100%
Preparation 4: Methyl (4R)-4-amino-5-phenylpentanoate
Stage 1: Methyl (3R)-3-[(tert-butoxycarbonyl)amino]-3-phenylpropanoate
1.9 ml of trimethylsilyldiazomethane as a 2.0M solution in cyclohexane are added to a solution of 0.31 g of (3R)-3-[(tert-butoxycarbonyl)amino]-3-phenylpropanoic acid, prepared according to the method described in Tetrahedron, 1997, 53, 12867-12874, in a mixture of 6 ml of ethyl ether and 6 ml of methanol. The reaction medium is stirred for 1 hour at room temperature and then hydrolysed with 100 μl of acetic acid and concentrated under reduced pressure. The crude product is triturated in a cyclohexane/ethyl acetate mixture (½) and filtered to give 0.386 g of the desired product.
Yield: 77%
NMR 1H (DMSO) δ (ppm): 7.40 (m, 1H), 7.30 (m, 5H), 4.40 (m, 1H), 3.60 (s, 3H), 2.30 (m, 2H), 1.80 (m, 2H), 1.40 (s, 9H)
MS: MH+294
HPLC: 100%
Stage 2: Methyl (4R)-4-amino-5-phenylpentanoate
1.62 ml (10 equivalents) of a 2.1M solution of hydrochloric acid in methanol are added to a solution of 0.1 g of the product obtained in stage 1 in 2.0 ml of methanol at 0° C. The reaction medium is stirred for 1 hour at room temperature and concentrated under reduced pressure to give 73 mg of the desired product.
Yield: 93%
1H NMR (DMSO) δ (ppm): 8.90 (m, 3H), 7.40 (m, 5H), 4.40 (m, 1H), 3.60 (s, 3H), 2.30 (m, 2H), 2.0 (m, 2H)
MS: MH+194
Preparation 5: (4-Cyclohexyl)phenylboronic acid
Stage 1: 4-[4-(Pinacolboro)phenyl]cyclohexyl
0.382 g of bis(pinacolato)diboron (1.2 equivalents), 27.5 mg of dichloro[[1,1′]-bis(diphenylphosphino)ferrocene]palladium(II) (0.03 equivalent), 41.7 mg of [1,1′]bis(diphenylphosphino)ferrocene (0.06 equivalent) and 0.369 g of potassium acetate (3 equivalents) are added, under nitrogen, to a solution of 0.3 g of bromophenylcyclohexyl in 5 ml of degassed 1,4-dioxane. The reaction medium is stirred for 17 hours at 80° C., diluted with ethyl acetate (100 ml), washed with water (3×60 ml), dried over sodium sulphate and concentrated under reduced pressure. Chromatography of the residue on silica gel (95/5 heptane/ethyl acetate) allows 0.2 g of the desired product to be obtained.
Yield: 56%
1H NMR (DMSO) δ (ppm): 7.60 (d, 2H), 7.20 (d, 2H), 1.70 (m, 5H), 1.40 (m, 4H), 1.30 (m, 12H)
Stage 2: (4-Cyclohexyl)phenylboronic acid
3 ml of water and 0.23 g of sodium periodate (3.0 equivalents) are added to a solution of 0.103 g of the compound obtained in stage 1 above in 3 ml of acetone. The reaction medium is stirred for 17 hours at 60° C. and then concentrated under reduced pressure. The residue obtained is dissolved in ethyl acetate (20 ml), washed with 1.0M hydrochloric acid solution and then with water (3× ml), dried over sodium sulphate, filtered and then concentrated under reduced pressure. Chromatography of the residue on silica gel (90/10 dichloromethane/methanol) allows 64 mg of the desired product to be obtained.
Yield: 87%
MS: MH−(+HCO2H) 249
HPLC: 100%
Preparation 6: Methyl 4-(4-bromophenyl)thiophene-2-carboxylate
Stage 1: Methyl 4-(4-nitrophenyl)thiophene-2-carboxylate
The product (1.94 g) is obtained according to the process of stage 1 of preparation 1, using methyl 4-bromothiophene-2-carboxylate and (4-nitrophenyl)boronic acid as substrates.
Yield: 78%
1H NMR (CDCl3) δ (Ppm): 3.92 (s, 3H), 7.75 (d, 2H), 7.82 (s, 1H), 8.12 (s, 1H), 8.30 (d, 2H)
Stage 2: Methyl 4-(4-aminophenyl)thiophene-2-carboxylate
A solution of 1.94 g of the compound obtained in stage 1 above in 20 ml of methanol containing 194 mg of 10% palladium-on-charcoal is stirred in an autoclave for 6 hours at 50° C. under 10 bar of hydrogen. The reaction medium is then filtered through Celite and concentrated under reduced pressure to give 1.51 g of the desired product.
Yield: 88%
1H NMR (DMSO) δ (ppm): 3.82 (s, 3H), 5.22 (s, 2H), 6.60 (d, 2H), 7.42 (d, 2H), 7.90 (s, 1H), 8.05 (s, 1H)
MS: MH+234
Stage 3: Methyl 4-(4-bromophenyl)thiophene-2-carboxylate
0.6 ml of concentrated hydrobromic acid is added to a solution of 103 mg of the product obtained in stage 2 above in 1.5 ml of water. The reaction medium is cooled to 0° C. and a solution of 35.5 mg of sodium nitrite (1.1 equivalents) in 0.5 ml of water is then added dropwise. After stirring for 1 hour at 0° C., a solution of 68 mg of copper bromide in 0.5 ml of concentrated hydrobromic acid is added dropwise. The reaction medium is stirred for a further 1 hour at 0° C. and then diluted with ethyl acetate (30 ml), washed with water (3×15 ml), washed with saturated sodium hydrogen carbonate solution (15 ml) and then washed again with water (15 ml). The organic phase is dried over sodium sulphate, filtered and then concentrated under reduced pressure. Chromatography of the residue on silica gel (95/5 cyclohexane/ethyl acetate) allows 52 mg of the desired product to be isolated.
Yield: 40%
1H NMR (CDCl3) δ (Ppm): 3.92 (s, 3H), 7.45 (d, 2H), 7.55 (d, 2H), 7.65 (s, 1H), 8.05 (s, 1H)
HPLC: 91.4%
246 mg of ethyl (2S)-amino(phenyl)acetate hydrochloride (1.1 equivalents), 395 mg of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) and 362 μl of N-ethyl-N,N-diisopropylamine are added to a solution of 300 mg of the compound obtained in Preparation 1 in 6 ml of anhydrous dimethylformamide. The reaction medium is stirred for 17 hours at room temperature and then hydrolysed. The precipitate formed is filtered off, washed with water and finally dried overnight to give 639 mg of the expected product.
Yield: 100%
1H NMR (DMSO) δ (ppm): 9.20 (d, 1H), 8.5 (s, 1H), 8.20 (s, 1H), 7.85 (d, 2H), 7.45 (m, 7H), 5.6 (d, 1H), 4.15 (m, 2H), 1.15 (t, 3H).
HPLC: 98.50%
170 mg of lithium hydroxide (5 equivalents) and 200 R1 of dimethylformamide are added to a solution of 639 mg of the compound obtained in Example 1 in 22 ml of an ethanol/water mixture (1/1). The reaction medium is stirred overnight at room temperature and then concentrated under reduced pressure. The solid obtained is taken up in water and acidified with 1.0M hydrochloric acid solution to pH 1. The precipitate formed is then filtered off, washed with water and then dried overnight to give 377 mg.
Yield: 64%
1H NMR (DMSO) δ (ppm): 13.0 (bs, 1H), 8.94 (s, 1H), 8.49 (s, 1H), 8.17 (s, 1H), 7.84 (d, 2H), 7.43 (m, 7H), 5.44 (s, 1H)
MS: MH+422
HPLC: 98.4%
The product (200 mg) is obtained according to the process of Example 1, using ethyl (2R)-amino(phenyl)acetate hydrochloride as substrate.
Yield: 44%
MS: MH+436
HPLC: 98,96%
The product (121 mg) is obtained according to the process of Example 2, using the compound obtained in Example 3 as substrate.
Yield: 65%
1H NMR (DMSO) δ (ppm): 13.01 (bs, 1H), 9.075 (d, 1H), 8.54 (s, 1H), 8.19 (s, 1H), 7.83 (d, 2H), 7.42 (m, 7H), 5.57 (d, 1H)
MS: MH+422
HPLC: 99.0%
The product (283 mg) is obtained according to the process of Example 1, using ethyl amino(phenyl)propanoate hydrochloride as substrate.
Yield: 59%
MS: MH+464
HPLC: 96.69%
The product (198 mg) is obtained according to the process of Example 2, using the compound obtained in Example 5 as substrate.
Yield: 75%
1H NMR (DMSO) δ (ppm): 9.03 (d, 1H), 8.29 (s, 1H), 8.15 (s, 1H), 7.82 (d, 2H), 7.46 (d, 2H), 7.41 (d, 2H), 7.34 (t, 2H), 7.25 (t, 1H), 5.40 (q, 1H), 2.83 (m, 2H)
MS: MH+436
HPLC: 99.3%
The products (60.9 mg and 50.2 mg) are obtained from the racemic mixture of Example 6 after a chiral separation on preparative HPLC under the following conditions:
Yield: 31%
1H NMR (DMSO) δ (ppm): 9.16 (bs, 1H), 8.29 (s; 1H), 8.14 (s, 1H), 7.82 (d, 2H), 7.45 (d, 2H), 7.40 (d, 2H), 7.33 (t, 2H), 7.24 (t, 1H), 5.38 (q, 1H), 2.79 (m, 2H)
MS: MH+436
HPLC: 99.21%
Yield: 26%
1H NMR (DMSO) δ (ppm): 12.51 (bs, 1H), 9.11 (m, 1H), 8.29 (s, 1H), 8.14 (s, 1H), 7.82 (d, 2H), 7.46 (d, 2H), 7.41 (d, 2H), 7.33 (t, 2H), 7.24 (t, 1H)
MS: MH+436
HPLC: 99.70%
A large excess of 28% aqueous ammonia solution is added, at 0° C., to a solution of 335 mg of the compound obtained in Example 8 in 5 ml of anhydrous tetrahydrofuran. The ice bath is removed and the mixture is stirred overnight at room temperature. The crude reaction mixture is concentrated under reduced pressure, hydrolysed and extracted with dichloromethane. The organic phases are combined, dried over sodium sulphate and concentrated. Chromatography of the residue on silica gel (70/30: cyclohexane/ethyl acetate) allows 33 mg of the desired product to be obtained.
Yield: 15%
1H NMR (DMSO) δ (ppm): 9.11 (d, 1H), 8.29 (s, 1H), 8.14 (s, 1H), 7.82 (d, 2H), 7.46 (m, 2H), 7.41 (m, 5H), 7.20 (m, 1H), 6.80 (m, 1H), 5.40 (m, 1H), 2.70 (m, 2H)
MS: MH−433
HPLC: 100%
The product (265 mg) is obtained according to the process of stage 1 of Preparation 2, using the compounds obtained in Preparations 1 and 2 as substrates.
Yield: 56%
1H NMR (DMSO) δ (ppm): 9.11 (m, 1H), 8.70 (s, 1H), 8.30 (m, 1H), 8.20 (s, 1H) 7.82 (d, 2H), 7.46 (m, 5H), 7.30 (m, 2H), 5.70 (d, 2H), 3.40 (m, 1H), 1.80 (m, 1H), 1.70 (m, 3H), 1.5 (m, 1H), 1.20 (m, 5H)
MS: MH+503
HPLC: 100%
The product (191 mg) is obtained according to the process of Example 1, using the product of Preparation 3 as substrate.
Yield: 29%
1H NMR (DMSO) δ (ppm): 8.70 (m, 1H), 8.20 (s, 1H), 8.10 (s, 1H), 7.82 (d, 2H), 7.46 (m, 2H), 7.30 (m, 5H), 4.70 (m, 1H), 4.20 (q, 2H), 2.90 (m, 2H), 2.60 (m, 2H), 1.0 (t, 3H)
MS: MH+478
HPLC: 100%
The product (104 mg) is obtained according to the process of Example 2, using the compound obtained in Example 11 as substrate.
Yield: 50%
1H NMR (DMSO) δ (ppm): 8.90 (m, 1H), 8.20 (m, 2H), 7.82 (d, 2H), 7.46 (m, 2H), 7.30 (m, 5H), 4.40 (m, 1H), 2.90 (m, 2H), 2.60 (m, 2H)
MS: MH−448
HPLC: 100%
The product (21.3 mg) is obtained according to the process of Example 1, using the product of Preparation 4 as substrate.
Yield: 15%
1H NMR (DMSO) δ (Ppm): 8.90 (m, 1H), 8.40 (s, 1H), 8.10 (s, 1H), 7.82 (d, 2H), 7.46 (m, 1H), 7.40 (m, 6H), 7.20 (m, 1H), 4.90 (m, 1H), 3.50 (s, 3H), 2.40 (m, 2H), 2.20 (m, 2H)
MS: MH+464
HPLC: 100%
The product (173 mg) is obtained according to the process of Example 2, using the compound obtained in Example 13 as substrate.
Yield: 73%
1H NMR (DMSO) δ (ppm): 8.90 (m, 1H), 8.40 (s, 1H), 8.10 (s, 1H), 7.82 (d, 2H), 7.46 (m, 2H), 7.36 (m, 5H), 4.90 (m, 1H), 2.40 (m, 2H), 2.20 (m, 2H)
MS: MH+450
HPLC: 98.9%
The product (0.155 g) is obtained successively according to the process of stage 1 of Preparation 1, using the product of Preparation 6 and of Preparation 5 as substrate, and then the process of Example 2, using the product obtained in the preceding stage as substrate, and then the process of Example 1, using ethyl (2S)-amino(phenyl)acetate as substrate.
Yield: 55,4%
1H NMR (DMSO) δ (ppm): 9.20 (m, 1H), 8.40 (s, 1H), 8.10 (s, 1H), 7.70 (d, 2H), 7.46 (m, 5H), 7.30 (m, 2H), 5.60 (d, 1H), 3.90 (s, 3H), 1.80 (m, 5H), 1.40 (m, 6H)
MS: MH+434
HPLC: 100%
The product (37.4 mg) is obtained according to the process of Example 2, using the compound obtained in Example 15 as substrate.
Yield: 25%
1H NMR (DMSO) δ (ppm): 12.9 (m, 1H), 9.10 (m, 1H), 8.40 (s, 1H), 8.10 (s, 1H), 7.82 (d, 2H), 7.46 (m, 5H), 7.30 (m, 2H), 5.50 (m, 1H), 1.80 (m, 5H), 1.40 (m, 6H)
MS: MH+420
HPLC: 100%
Stage 1: Methyl 4-[4-(4-acetyl-phenyl)phenyl]-thiophene-2-carboxylate
The product (1.73 g) is obtained according to the process of stage 1 of Preparation 1, using the product of Preparation 6 and (4-acetyl-phenyl)boronic acid as substrates.
Yield: 76%
MS: MH+337
Stage 2: 4-[4-(4-Acetyl-phenyl)phenyl]-thiophene-2-carboxylic acid
The product (1 g) is obtained according to the process of Example 2, using the product obtained in stage 1 above as substrate.
Yield: 61%
MS: MH+323
Stage 3: Ethyl (2S)-({4-[4-(4-Acetyl-phenyl)phenyl]thien-2-yl}carboxamido)-(phenyl)acetate
The product (360 mg) is obtained according to the process of Example 1, using ethyl (2S)-amino(phenyl)acetate hydrochloride as substrate.
Yield: 44%
1H NMR (CDCl3) δ (Ppm): 9.20 (d, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 8.05 (d, 2H), 7.90 (d, 2H), 7.85 (s, 4H), 7.50 (m, 2H), 7.40 (m, 3H), 5.60 (d, 1H), 4.20 (m, 2H), 2.65 (s, 3H), 1.20 (t, 3H)
The product (16 mg) is obtained according to the process of Example 2, using the compound obtained in Example 17 as substrate.
Yield: 5%
1H NMR (DMSO) δ (ppm): 13.0 (bs, 1H), 9.10 (d, 1-H), 8.60 (s, 1H), 8.25 (s, 1H), 8.05 (d, 2H), 7.90 (m, 6H), 7.50 (m, 2H), 7.40 (m, 3H), 5.55 (d, 1H), 2.60 (s, 3H)
MS: MH+456
HPLC: 95.9%
The product (1.012 g) is obtained according to the process of Example 17, replacing the ethyl (2S)-amino(phenyl)acetate hydrochloride with ethyl (2R)-amino(phenyl)propanoate hydrochloride.
Yield: 66%
1H NMR (DMSO) δ (ppm): 9.10 (d, 1H), 8.30 (s, 1H), 8.25 (s, 1H), 8.15 (d, 2H), 7.90 (m, 6H), 7.35 (m, 5H), 5.40 (m, 1H), 4.10 (q, 2H), 3.0 (m, 2H), 2.60 (s, 3H), 1.30 (t, 3H)
MS: MH+498
HPLC: 97.7%
The product (0.641 g) is obtained according to the process of Example 2, using the compound obtained in Example 19 as substrate.
Yield: 91%
1H NMR (DMSO) δ (ppm): 9.10 (d, 1H), 8.3 (s, 1H), 8.25 (s, 1H), 8.15 (d, 2H), 7.90 (m, 6H), 7.35 (m, 5H), 5.40 (m, 1H), 3.0 (m, 2H), 2.60 (s, 3H)
MS: MH+470
HPLC: 100%
Stage 1: Methyl 4-[4-(pyrid-4-yl)phenyl]thiophene-2-carboxylate hydrochloride
The crude product (60 g) is obtained in the form of a yellow solid by applying the process of stage 1 of Preparation 1, using the product of Preparation 6 and (4-pyridyl)boronic acid as substrates. The organic phase obtained after extraction is acidified with 2M hydrochloric acid solution to precipitate out the desired product, which is finally isolated by filtration.
1H NMR (DMSO) δ (ppm): 8.91 (d, 2H), 8.50 (s, 1H), 8.38 (s, 1H), 8.36 (d, 2H), 8.10 (d, 2H), 8.06 (d, 2H), 3.45 (s, 3H)
Stage 2: 4-[4-(4-Acetylphenyl)phenyl]thiophene-2-carboxylic acid hydrochloride
The product (19.4 g) is obtained according to the process of Example 2, using the product obtained in stage 1 above as substrate.
Yield for stages 1 and 2: 44.5%
1H NMR (DMSO) δ (ppm): 8.88 (d, 2H), 8.43 (s, 1H), 8.30 (d, 2H), 8.28 (s, 1H), 8.08 (d, 2H), 8.02 (d, 2H)
Stage 3: Ethyl (2R)-3[(({4-[4-(4-acetylphenyl)phenyl]thien-2-yl}carboxamido)-(phenyl)propanoate
The product (1.43 g) is obtained according to the process of Example 1, using ethyl (2R)-amino(phenyl)propanoate hydrochloride as substrate.
Yield: 100%
1H NMR (DMSO) δ (ppm): 8.15 (d, 1H), 8.76 (d, 2H), 8.35 (s, 1H), 8.22 (s, 1H), 7.95 (d, 2H), 7.90 (d, 2H), 7.88 (d, 2H), 7.44 (d, 2H), 7.35 (t, 2H), 7.26 (t, 1H), 5.42 (q, 1H), 4.05 (q, 2H), 2.95 (m, 2H), 1.10 (t, 3H)
HPLC: 100%
The product (0.990 g) is obtained according to the process of Example 2, using the compound obtained in Example 21 as substrate.
Yield: 68%
1H NMR (DMSO) δ (ppm): 9.02 (d, 1H), 8.88 (d, 2H), 8.46 (s, 1H), 8.34 (d, 2H), 8.30 (s, 1H), 8.11 (d, 2H), 7.95 (d, 2H), 7.44 (d, 2H), 7.35 (t, 2H), 7.26 (t, 1H), 5.43 (q, 1H), 2.89 (m, 2H)
HPLC: 100%
The product (17.8 mg) is obtained according to the process of Example 1, using the compound obtained in Preparation 7 as substrate.
Yield: 23%
1H NMR (DMSO) δ (ppm): 9.10 (d, 1H), 8.50 (s, 1H), 8.25 (s, 1H), 7.80 (d, 2H), 7.40 (m, 12H), 5.90 (m, 1H), 4.40 (m, 4H)
MS: MH+526
HPLC: 100%
in vitro Evaluation of the Inhibitory Activity of the Compounds of the Invention on MMP-12:
The inhibitory activity of the compounds of formula (I) on metalloprotease-12 is evaluated by testing the capacity of the compounds of the invention to inhibit the proteolysis of a peptide that is an MMP-12 substrate.
The substrate peptide used (fluorigenic peptide-1: FP-1) in the test has the following sequence: Mca-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2.
The inhibitory activity of a compound of formula (I) is expressed as the IC50 value, which represents the concentration of inhibitor for which a 50% inhibition of the metalloprotease is observed.
The reaction starts with the sequential addition of 41 μl of FP-1 substrate (final concentration of 10 μM) to a buffer solution of 50 mM of Tris-HCl and 10 mM of CaCl2, and containing 5 mM of hydroxamic acid and 5 μl of the enzyme diluted in a 0.005% Brij-35 buffer solution. The microplates are incubated for 20 minutes at room temperature. The compounds of the invention are tested at concentrations ranging from 0.3 to 30 μM. The measurement of the amount of proteolysis of the peptide substrate is monitored by means of a measurement of absorbance at 405 nm using a microplate spectrophotometer, at room temperature. The IC50 values are calculated from curves in which the percentage of the catalytic activity relative to the control is represented on the x-axis and the inhibitor concentration is represented on the y-axis.
The test described above for the inhibition of MMP-12 is adapted and used to determine the capacity of the compounds of formula (I) to inhibit the metalloproteases MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13 and MMP-14. The results obtained show that the compounds of the invention generally have IC50 values for MMP-12 that are from 5 to more than 100 times lower than the IC50 values obtained for the same compound with the other metalloproteases tested, thus proving their capacity for selective inhibition with respect to metalloprotease-12 (MMP-12). More specifically, the compounds of the present invention generally show selectivity with a factor of greater than 50 towards the metalloproteases mentioned above, except with regard to MMP-13. Thus, the compounds of the present invention also show inhibitory activity on MMP-13, also allowing the use of the pharmaceutical compositions containing one or more compounds of the invention for the treatment of pathologies associated with an activity of MMP-13. Among these pathologies that may be mentioned, as a guide and with no limitation, are cancer, osteoporosis, osteoarthritis, arthritis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, cardiac insufficiency, asthma and chronic obstructive bronchopneumopathy.
By way of example and with no limitation of the invention, the table shows a number of results of activity of the compounds of the invention with respect to MMP-12 and MMP-13.
| Number | Date | Country | Kind |
|---|---|---|---|
| 03291641.3 | Jul 2003 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| 60494654 | Aug 2003 | US |
